The survival of any species demands the faithful inheritance of genetic information. Essential to this process are the directed movements and positioning of chromosomes or plasmids such that they are accurately distributed to the daughter cells at cell division. Although prokaryotes do not undergo the complex mitotic steps associated with eukaryotic cells, prokaryotic chromosomes are nevertheless dynamically arranged during the cell cycle via the action of segregation, or par systems. Coordination of this process requires precise protein-DNA and protein-protein interactions carried out by par systems. All known plasmid encoded par loci specify three components: a cis-acting centromere site (parS), and two proteins, ParB and ParA. In the E. coli P1 par system, which has served as a paradigm for understanding partition, the genes for ParA and ParB form an operon, and the approximately 74 bp partition site, parS, is located immediately downstream of parB . ParB, a 38 kDa protein with no sequence homology to any protein, is a DNA-binding protein and, along with IMF or alone, binds to a highly complex centromere-like site, parS, to form the partition complex. ParA, a 44 kDa Walker-type ATPase, utilizes the energy of ATP hydrolysis to drive plasmid separation after interacting with ParB in the partition complex. Our recent structure determinations of ParB and the minimal partition site have revealed novel DNA-binding characteristics of ParB that explain its ability to bind, spread and pair plasmids. Thus, these structures have provided unprecedented insight into the mechanism of partition complex formation. Completely unknown, however, are structural bases for plasmid separation, the step carried out by ParA. Thus, in this proposal we will build on our recent progress towards a full elucidation of P1 partition with the following Specific Aims: (1) fully elucidate the mechanism of P1 partition complex formation through structural and biochemical studies on the ParB-parS partition complex. (2) Clarify the mechanism of P1 plasmid separation via structural and biochemical studies on the key end states of P1 ParA (apoParA, ParA-ADP and the ParA(K188Q)-(AMP-PCP)-ParB(1-28) complex). These structures will provide the foundation for understanding how partition systems function to drive chromosome segregation in prokaryotes and may provide potential points of therapeutic intervention against pathogenic bacteria, which also depend on par systems for segregation and thus, survival.